Normally, cells have mechanisms to control growth and proliferation, such that a cell will only divide and grow under certain circumstances, and in a controlled manner. Cells also have mechanisms that induce cell death, or apoptosis, when the normal regulatory mechanisms that govern cell growth and proliferation are subverted. However, in some instances, such proliferation control and apoptotic mechanisms breakdown, allowing the cell to proliferate unchecked, potentially leading to a proliferative disorder or disease within an organism. Such proliferative disorders include cancer and other hypercellular lesions such as psoriasis, warts and keloids.
Uncontrolled cellular proliferation may result from dysregulation of gene expression. Cells have a tightly controlled cycle of DNA replication and division (the cell cycle), which is regulated by a series of cell cycle and transcription factors. One set of cell cycle transcription factors, the E2F transcription factors, have been shown to play a crucial role in regulating cellular proliferation by integrating the activity of the cell cycle machinery with that of the transcriptional apparatus in a manner that contributes to the timely expression of genes required for cell cycle progression and proliferation (Lam, E., et al., Curr Opin Cell Biol 6, 859-866 (1994); Dyson, N. (1998), Genes Dev 12: 2245-2262; Helin, K, (1998), Curr Opin Genet Dev 8: 28-35; Harbour, J. W., and Dean, D. C. (2000), Genes Dev 14: 2393-2409; Trimarchi, J. M. and Lees, J. A. (2002), Nat Rev Mol Cell Bio 3: 11-20). The E2F transcription factors, in particular E2F-1, have been implicated in the regulation of apoptosis (Muller, H., et al., Genes Dev 15, 267-285 (2001); Nahle, Z., et al., Nat Cell Biol, (2002) 11:859-64; Philips, A. C., and Vousden, K. H. Apoptosis 6, 173-182 (2001)).
Cyclin E-associated kinase activity has been shown to be essential for traversing the restriction point and executing S phase entry during cell cycle progression (Keyomarsi, K., and Herliczek, T. W. (1997), Prog Cell Cycle Res 3: 171-191). The entry into, passage through and exit from the cell cycle are precisely controlled by the sequential activation of the cyclin-dependent kinases (Cdks) Cdk4/6, Cdk2 and Cdc2 (Sherr, C. J., and Roberts, J. M. (1999), Genes Dev 13: 1501-1512). Commitment of cells to enter the cell cycle is regulated by growth factors (both positive and negative) that exert their effect during the G1 phase.
The G1 D-type cyclins, in conjunction with cyclin-dependent kinases Cdk4 and Cdk6, play key roles in the execution of mitogen-induced cellular proliferation. Positive (mitogenic) growth factors convey growth-stimulatory signals that promote the synthesis of the D-type cyclins and their assembly into active Cdk4/6-cyclin D complexes, resulting in their catalytic activation and substrate recognition. Active Cdk4/6-cyclin D complexes contribute to cyclin-E/Cdk2 activation and generation of hypo-phosphorylated RB. The hypo-phosphorylated RB bound to transcriptionally inactive E2F-1/DP-1 complexes serves as a target for hyper-phosphorylation by activated cyclin-E/Cdk2, resulting in the dissociation of RB, derepression/activation of E2F-responsive genes and release of cells from the late G1 checkpoint (Sherr, C. J. (1993), Cell 73: 1059-1065; Lam et al., supra; Dyson, supra; Helin, supra; Harbour, J. W., et al., (1999), Cell 98: 859-869; Sherr, (1999), supra; Harbour (2000), supra; Trimarchi et al., supra).
As key regulators of mitogenic-signaling pathways, Cdk4/Cdk6-cyclin D activities are regulated at multiple levels, including the synthesis and binding of cyclin D, both inhibitory and activating phosphorylation events, and the association/dissociation of inhibitory molecules called Cdk inhibitors (CKIs) (Sherr (1999), supra). Cdk proteins are inactive unless they are bound to the respective cyclin partners. Upon binding, each cyclin-Cdk complex is further subjected to negative and positive regulation through specific phosphorylations. Further negative regulation is provided by the binding of specific Cdk inhibitors (CKI) that disrupt the active site and interfere with ATP binding. Mammalian CKIs are classified into two families, the Cip/Kip (Cdk2/kinase inhibitor protein) and the Ink4 (inhibitors of cyclin-dependent kinase 4) inhibitors, both of which restrain cell cycle progression by specifically and coordinately binding to Cdk complexes controlling G1 and G1/S phases. While the Cip/Kip family CKIs are less specific, inhibiting Cdk2, Cdk4 and Cdk6 activities, the Ink4 inhibitors (p16/Ink4a, p15/Ink4b, p18/Ink4c and p19/Ink4d) bind specifically to cyclin D-Cdk4 and -Cdk6 complexes.
An additional level of regulation imposed on Cdk4 has recently been identified. p34SEI-1, a novel cyclin-dependent kinase 4 (Cdk4)-binding protein that appears to antagonize the function of p16INK4a, has been shown to render the cyclin D1-Cdk4 complex resistant to inhibition by p16INK4a (Sugimoto, M., et al., (1999), Genes Dev 13: 3027-3033). p34SEI-1 is a serum inducible protein whose ectopic expression enables fibroblasts to proliferate even in low serum concentrations. p34SEI-1 has been proposed to play key functional role(s) in facilitating the formation and activation of cyclin D-Cdk complexes and mediating mitogen-driven cell cycle progression (Sugimoto et al., supra).
In order to address proliferative disorders that result when the regulation of the above-described pathway of cell cycle progression is rendered abnormal, it is important to find ways of blocking cell cycle progression in abnormally proliferating cells. Thus, there exists a need for novel treatments and methods of inducing proliferative block in cells.